QC summary of the EpiPlex assay
Requirements for Read QC check: - Total Reads ≥ 10000000 - Insert Size ≥ 125 - Percent Clean Reads ≥ 90 - Percent Duplicates Genome ≤ 50 Requirements for Peak QC check: - m6A Read Count ≥ 6000000 - Ino Read Count ≥ 1000000 - m6A Percent DRACH Peaks ≥ 80 - Ino Percent A-to-I Editing Peaks ≥ 85 Requirements for Spike-In QC check: - Ino Enr Ratio ≥ 1 - m6A Enr Ratio ≥ 1Quality of reads before peak calling
Number of usable reads per modification. Figures represent number of reads used by the peak caller.
Total and final read counts used by the peak caller.
Read Counts: Number of mapped reads associated with each modification. Unique Mapped Read Count: number of deduplicated mapped reads associated with each modification. FRIP: fraction of unique reads used for peak calling. Useful for knowing efficiency of the EpiPlex assay.
Quality of m6A and inosine peak calling.
General metrics of peaks called by EpiScout.
Peaks: number of peaks called by the peak caller passing filtering criteria. Genomic coverage: Sum of called peak widths. Useful for knowing magnitude of modification density in a sample. Peak width: Mean peak width of peaks in a sample. Useful for knowing the typical size of a modified region.
Metrics related to a modification's representative motif (DRACH for m6A, A-to-I editing site for Inosine).
Percent motifs in peaks: Percent of peaks containing at least one relevant motif. Useful for validation of m6A peaks with DRACH, or validation of inosine peaks with an A-to-I event. Motif per kb: Density of motifs found within called peaks. Useful for determining motif enrichment of called peaks. Higher density indicates higher modification levels.
Relative abundance of peaks found at transcript locations.
m6A Peak Locations: Normalized abundance of m6A modifications in annotated genomic regions. m6A is typically enriched in the exonic and three prime UTR regions. Ino Peak Locations: Normalized abundance of inosine modifications in annotated genomic regions. Inosine is typically enriched in intronic and intergenic regions.
Distribution of peaks along a hypothetical transcript binned to 100 positions. 3'UTR and 5'UTR positions are experimentally determined, and denoted at bins 10 and 60.
Metagene Distribution: m6A is expected to be enriched at the exon-3'UTR junction, while inosine is generally distributed throughout the coding segment.
RiP Correlation Plots: Reads In Peaks correlations for Enriched and Solution Control pileups. Pileups are scaled according to spike-in control and genome size.
Correlation Values: R-squared values of enrichment and solution control scaled RiP.
Upset Plot: Intersections of peaks common across samples, similar to a venn diagram. Vertical bars represent number of peaks common to samples joined by dots below. Horizontal bars represent total number of peaks per sample.
RiP Correlation Plots: Reads In Peaks correlations for Enriched and Solution Control pileups. Pileups are scaled according to spike-in control and genome size.
Correlation Values: R-squared values of enrichment and solution control scaled RiP.
Upset Plot: Intersections of peaks common across samples, similar to a venn diagram. Vertical bars represent number of peaks common to samples joined by dots below. Horizontal bars represent total number of peaks per sample.
RiP Correlation Plots: Reads In Peaks correlations for Enriched and Solution Control pileups. Pileups are scaled according to spike-in control and genome size.
Correlation Values: R-squared values of enrichment and solution control scaled RiP.
Upset Plot: Intersections of peaks common across samples, similar to a venn diagram. Vertical bars represent number of peaks common to samples joined by dots below. Horizontal bars represent total number of peaks per sample.
RiP Correlation Plots: Reads In Peaks correlations for Enriched and Solution Control pileups. Pileups are scaled according to spike-in control and genome size.
Correlation Values: R-squared values of enrichment and solution control scaled RiP.
Upset Plot: Intersections of peaks common across samples, similar to a venn diagram. Vertical bars represent number of peaks common to samples joined by dots below. Horizontal bars represent total number of peaks per sample.
RiP Correlation Plots: Reads In Peaks correlations for Enriched and Solution Control pileups. Pileups are scaled according to spike-in control and genome size.
Correlation Values: R-squared values of enrichment and solution control scaled RiP.
Upset Plot: Intersections of peaks common across samples, similar to a venn diagram. Vertical bars represent number of peaks common to samples joined by dots below. Horizontal bars represent total number of peaks per sample.
RiP Correlation Plots: Reads In Peaks correlations for Enriched and Solution Control pileups. Pileups are scaled according to spike-in control and genome size.
Correlation Values: R-squared values of enrichment and solution control scaled RiP.
Upset Plot: Intersections of peaks common across samples, similar to a venn diagram. Vertical bars represent number of peaks common to samples joined by dots below. Horizontal bars represent total number of peaks per sample.
RiP Correlation Plots: Reads In Peaks correlations for Enriched and Solution Control pileups. Pileups are scaled according to spike-in control and genome size.
Correlation Values: R-squared values of enrichment and solution control scaled RiP.
Upset Plot: Intersections of peaks common across samples, similar to a venn diagram. Vertical bars represent number of peaks common to samples joined by dots below. Horizontal bars represent total number of peaks per sample.
